Regulation and function of thioredoxin reductase 1 in human tumor cells

Abstract: The thioredoxin system is a general protein disulfide reductase system present in all organisms. Its function is to maintain an intracellular reducing environment and upholding a defense against reactive oxygen species generated as by-products of aerobic metabolism. The constituents of the system are Thioredoxin (Trx), Thioredoxin reductase (TrxR1) and NADPH. Apart from its central position as an antioxidant protection and redox regulatory system it interacts in diverse cellular processes, such as growth regulation, proliferation, apoptosis and various signaling pathways. The system is represented by different izoenzymes in different cellular localizations. The aim of this thesis was to study the mainly cytosolic variant TrxR1 in regards to regulation and function in human tumor cells. TrxR1 is a selenium-containing protein with catalytic activity entirely dependent on the incorporation of this element. The enzyme furthermore functions as the core metabolizing entity of different selenium compounds. The specific pathological condition chosen for the studies was lung cancer, a malignancy with exceptionally poor prognosis. The expression of mammalian TrxR1 is governed by tremendously complex mechanisms the purposes of which are not entirely known. The main aspect of this is the occurrence of extensive alternative splicing generating several protein isoforms. In humans, 21 different mRNA splice variants possibly encoding five protein isoforms was previously identified in database searches and cDNA libraries. This study set out to confirm the existence of a number of these mRNA variants and protein isoforms in human cells and investigate connections between expression profiles and cellular morphology. All investigated splice variations explored as well as two protein isoforms never before recognized in human cells could be identified and a significant correlation connecting expression of TrxR with different malignant phenotypes. In addition, the cytotoxic effects of the inorganic selenium compound sodium selenite in comparison to conventional chemotherapeutic drugs with special reference to the before mentioned regulation of TrxR was explored. In affirmation of previous findings, selenite in comparably moderate concentrations showed immense cytotoxic properties on drug resistant cell lines. These effects were further enhanced by inhibiton of TrxR. Furthermore, enzymatic inhibition with a novel gold compound was investigated as a possible strategy for sensitization in radiotherapy. Results showed that inhibition of TrxR, non-cytotoxic in itself, greatly decreased the ability of cancer cells to repopulate after subjection to radiation treatment. Finally, using immunohistochemistry on paraffin embedded tissue from lung cancer, the expression of thioredoxin family redox proteins in association to differentiation was examined. Several of these displayed significant correlation to both differentiation and proliferation, suggesting possible opportunities for diagnostic purposes. Taken together these results strongly support previous hypotheses of the importance of redox enzymes and TrxR1 in particular, in various aspects of cancer development and management.